Antoine Loquet

Structure determination of fully labeled proteins
by solid-state NMR
Antoine Loquet
IBCP-CNRS Lyon, France
a.loquet@ibcp.fr
. Distance restraints on fully 13C, 15N labeled proteins
. Handle highly ambiguous solid-state NMR data
. Application to high-resolution structure calculation of the Crh protein
. Additional restraints: JCHHC experiment
. Carbon-carbon correlation for 3D structure determination: TSAR
a.loquet@ibcp.fr
. Distance restraints on fully 13C, 15N labeled proteins
. Handle highly ambiguous solid-state NMR data
. Application to high-resolution structure calculation of the Crh protein
. Additional restraints: JCHHC experiment
. Carbon-carbon correlation for 3D structure determination: TSAR
a.loquet@ibcp.fr
Internuclear distance measurements ?
1
H ...1H
large dipolar coupling: broad lines in proton spin-diffusion exp.
Biochemical tricks: spin dilution (Reif / Zilm / Rienstra)
i.e. high-resolution structure of GB1 with back-exchanged1 H sample
Zhou et al., Angew. Chem. Int. Ed. 2008
a.loquet@ibcp.fr
Internuclear distance measurements ?
1
H ...1H
large dipolar coupling: broad lines in proton spin-diffusion exp.
Biochemical tricks: spin dilution (Reif / Zilm / Rienstra)
i.e. high-resolution structure of GB1 with back-exchange 1H sample
Zhou et al., Angew. Chem. Int. Ed. 2008
13
C ... C
dipolar truncation
Hohwy et al., J. Chem. Phys 2002
Grommek et al., Chem. Phys. Lett. 2006
Number of contacts
13
Loquet et al., J. Am. Chem. Soc 2008
300
150
0
1.5
a.loquet@ibcp.fr
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Carbon-carbon distance / Å
sequential
(|i-j|=1)
long-range
(|i-j|>4)
intraresidue
medium-range
(1<|i-j|<4)
6.0
6.5
7.0
Internuclear distance measurements ?
1
H ...1H
large dipolar coupling: broad lines in proton spin-diffusion exp.
Biochemical tricks: spin dilution (Reif / Zilm / Rienstra)
i.e. high-resolution structure of GB1 with back-exchange 1H sample
Zhou et al., Angew. Chem. Int. Ed. 2008
13
C ... C
dipolar truncation
Hohwy et al., J. Chem. Phys 2002
Grommek et al., Chem. Phys. Lett. 2006
few kHz coupling
Number of contacts
13
Loquet et al., J. Am. Chem. Soc 2008
300
150
0
1.5
2.0
2.5
-> 1-bond / 2-bonds dominate
a.loquet@ibcp.fr
3.0
3.5
4.0
4.5
5.0
5.5
Carbon-carbon distance / Å
sequential
(|i-j|=1)
long-range
(|i-j|>4)
intraresidue
medium-range
(1<|i-j|<4)
6.0
6.5
7.0
Pulse sequences for fully labeled system
. Spin diffusion, DARR, RAD
Franks et al., PNAS 2007
Manolikas et al., J. Am. Chem. Soc 2008
. Proton mediated, rare spin detected
Lange et al., Angew. Chem. Int. Ed. 2008 / Korukottu et al., PloS ONE 2008
Loquet et al., J. Am. Chem. Soc 2008-a / Loquet et al., J. Am. Chem. Soc 2008-b
H
C
H
C
H
C
H
C
H
. Third Spin Assisted Recoupling
De Paëpe et al., accepted
a.loquet@ibcp.fr
C
C
Proton mediated, rare spin detected: N/CHHC
10
Lange et al., J. Am. Chem. Soc 2002
Lange et al., J. Am. Chem. Soc 2003
!
2
1H
13C
CP
spinal64
CP
CP
t1
CP
15N
mixing
time
!
2
!
2
1H
!
2
CP
CP
13C
spinal64
t1
CP
20
CP
spinal64
CP
t2
!
2
mixing
time
CP
spinal64
CP
CP
t2
Carbon 13 chemical shift / ppm
!
2
30
40
50
60
70
60
40
20
Carbon 13 chemical shift / ppm
CHHC, mixing time: 200µs
at 500 MHz, 4 mm rotor
fully labeled Crh protein
a.loquet@ibcp.fr
Proton mediated, rare spin detected: N/CHHC
10
1. Intensity <-> distance ?
2. Enough information for
3D structure calculations ?
3. Deal with high-level of ambiguity
Carbon 13 chemical shift / ppm
20
30
40
50
60
70
60
40
20
Carbon 13 chemical shift / ppm
CHHC, mixing time: 200µs
at 500 MHz, 4 mm rotor
fully labeled Crh protein
a.loquet@ibcp.fr
12
0.8
12-13
43-44
62-63
65-66
26-27
47-48
48-49
51-52
77-78
H(i)
Cα
N
C
Cα
9
Signal intensity / a. u.
Peak intensity / a.u.
1. Build-up magnetization curves
Hα(i-1)
O
6
Cα
N
3
0
Hα(i-1)
H(i)
C
Cα
O
0
20
40
60
80
0.6
0.2
0
100
Mixing time / µs
<3Å
3-4 Å
>4Å
0.4
0
100
200
Proton-proton mixing time / µs
300
360
N
H
2.18 Å
H
Leu10
300
γ
χ2
240
HN
180
120
possible for simple case:
3.88 Å
Leu74
60
0
Peak intensity / a.u.
0.8
0.6
0.4
0.2
0
0
intraresidue
sequential
one H attached
Signal intensity / a. u.
1
60
120
180
χ1
240
300
360
Hγ
0
50
100
1H-1H mixing time / µs
150
L10 (2.18 Å)
L53 (4.60 Å)
L74 (3.88 Å)
6
an unique upper distance,
4
2
no distance classes for calculations
0
0
a.loquet@ibcp.fr
25
50
75
Mixing time / µs
100
Gardiennet, Loquet, Etzkorn, Heise, Baldus, Böckmann
J. Biomol. NMR 2008 40(4), 239-250
2. Assignment using back-calculated spectra
Carbon 13 chemical shift / ppm
20
40
60
80
54CB-55CB
47CD1-19CA
64CD1-35CA 44CB-77CB
64CD1-41CD
51CE-14CA
42CG2-40CE
64CD1-62CG2 51CE-19CB
64CD1-62CA
54CB-13CA
1CE-2CA
26CB-22CA
47CG1-48CG
33CG1-65CA
65CB-31CA
26CB-27CB
55CG1-54CA
61CG1-59CA
55CG2-54CB
53CG-52CB
41CG-35CB
74CG-63CB
37CD-59CA
17CB-18CD
40CD-39CA
45CD-44CA
64CG1-62CB
23CB-47CD1
68CB-31CB
45CD-31CA 60CB-59CA 60CG-59CA
66CG-31CB
47CB-50CB
66CB-65CA
61CB-62CA
22CB-50CB
75CG-78CB
36CB-62CB
42CB-44CA 36CB-35CB
41CE-64CB
55CB-54CA
47CB-20CB
47CB-46CB
29CB-73CA
45CE-29CB
29CB-30CA
22CB-44CB
29CB-30CB
77CB-63CB
45CE-27CB
35CB-53CG
32CB-31CA
37CE-59CA
13CA-55CB
64CB-65CA
32CB-31CB
14CB-51CE
63CB-61CB
13CA-14CB
63CB-35CA
58CA-57CB
63CB-78CB
63CB-62CB
35CB-42CG1
63CB-62CA
67CA-68CB
13CA-55CA
63CB-77CA
63CB-35CB
54CA-55CA
35CB-14CD1
49CA-42CB
49CA-51CA
69CA-67CA
60CA-62CA
65CA-66CG
44CA-23CG2
54CA-37CE
18CD-14CA
46CA-47CA
54CA-37CB
20CA-47CD1
34CA-35CB
56CA-59CA
40CA-39CA
69CA-73CA
69CA-68CB
14CA-18CA
56CA-13CA 46CA-47CG1
32CA-65CA
29CA-30CB
48CA-47CG1
25CA-23CA
74CA-63CB
45CA-44CA
48CA-51CE
25CA-22CA
62CA-64CG1
85CA-79CB
42CA-49CA
64CA-62CB
12CA-54CA
31CA-33CG2
52CA-49CA
68CA-31CB
30CA-45CD
22CA-23CA
31CA-33CA
31CA-67CA
55CA-59CA
81CA-79CB
18CA-14CG
31CA-32CA
31CB-30CB
62CA-36CB
31CA-30CA
59CA-58CA
23CA-45CD
47CA-46CB
30CA-68CG
31CB-33CA
31CB-33CB
59CB-58CA
30CB-31CA
62CB-61CB
47CA-51CA
31CA-29CA
18CA-22CB
59CB-55CA
31CA-45CE
30CA-29CA
80CA-77CB
62CB-61CA
52CB-40CE
81CA-77CA
18CA-14CB
80
40
60
20
Carbon 13 chemical shift / ppm
DARR (mixing: 200ms)
4 mm rotor
500 MHz
a.loquet@ibcp.fr
Simulated spectra to assign restraints
in CHHC, NHHC and DARR:
0.25 ppm tolerance
1 possible assignment
cutoff 5Å for H...H correlation
cutoff 7Å for C...C correlation
2. Assignment using back-calculated spectra
CC
HH
2.5 Å
2.5 Å
CC+HH
1.6 Å
+ TALOS
1.5 Å
a.loquet@ibcp.fr
1.4 Å
1.3 Å
Loquet, Bardiaux, Gardiennet, Blanchet, Baldus, Nilges, Malliavin, Böckmann
J. Am. Chem. Soc. 2008 130(11) 3579-3589
< 1 % cross-peaks are assignable
de novo structure determination ?
deal with high-level of ambiguity
2 problems
1. each cross-peak is highly ambiguous in its CS assignment
2. each cross-peak could be intra or/and intermonomeric
pdb entry: 1mu4
Juy et al., J. Mol. Biol. 2003
a.loquet@ibcp.fr
Deal with ambiguity
1. Ambiguity treatment
N
-6 -1/6
D = (! d )
ADR
(Ambiguous Distance Restraints)
Nilges, J. Mol. Biol. 1995
a=1
-6
-6
-1/6
D = ( d intra +dinter )
2. Asymmetric labeling
Weiss, J. Magn. Reson. 1990
Arrowsmith et al., Biochemistry 1990
application on heterogenously labeled Crh protein
Etzkorn et al., J. Am. Chem. Soc 2004
25 intermonomeric H...H restraints
Dimeric topology of microcrystals Crh protein:
A
B
AA and BB
AB and BA
2 ambiguities per cross-peak
a.loquet@ibcp.fr
Nilges, Proteins 1993
N
-6
-6
-1/6
D = (! d intra + !dinter )
a=1
with more than 10 ambiguities
for CS assignment
manual peak-picking on:
CHHC (200µs)
NHHC (100µs)
N¹⁵ chemical shift / ppm
Input data for ARIA calculation
100
a
120
140
70
20
Carbon 13 chemical shift / ppm
Talos dihedral angle restraints
no H bonds, homology model, etc...
50
40
30
20
Carbon 13 chemical shift / ppm
treated as ADR, one distance class 1.8-5.0Å
20 intermonomeric NHHC distance restraints
60
b
30
40
50
60
70
70
60
50
40
30
Carbon 13 chemical shift / ppm
Loquet et al., J. Am. Chem. Soc. 2008
a.loquet@ibcp.fr
20
Protocol
ARIA 2.2:
NCS
C2 symmetry
packing restraint
slow cooling
input
1st ARIA run
best conformers
template structures
2nd ARIA run
unambiguous assignments
xplor round
ss-NMR structures
a.loquet@ibcp.fr
1500
unambiguous
ambiguous
long-range
count
1200
900
600
300
0
0
1
2
3
4
5
6
7
8
iteration
14
12
precision
accuracy
rmsd / Å
10
8
6
4
1st run
2
0
0
1
2
3
4
5
6
7
8
iteration
rmsd / Å
8
precision
accuracy
6
4
2nd run
2
0
0
1
2
3
4
5
iteration
a.loquet@ibcp.fr
3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints
Loquet, Bardiaux, Gardiennet, Blanchet, Baldus, Nilges, Malliavin, Böckmann
J. Am. Chem. Soc. 2008 130(11) 3579-3589
ss-NMR structures
a.loquet@ibcp.fr
unambiguous distance restraints
total
sequential
medium-range
long-range
643
181
85
131
distance violations > 0.5 A
none
rmsd precision
monomer, backbone
dimer backbone
0.87 Å ± 0.12 Å
1.30 Å ± 0.20 Å
accuracy
monomer
dimer
1.62 Å
2.89 Å
3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints
Loquet, Bardiaux, Gardiennet, Blanchet, Baldus, Nilges, Malliavin, Böckmann
J. Am. Chem. Soc. 2008 130(11) 3579-3589
new methods for distance restraints
determination
(also for large fully labeled system)
!
2
!
2
1H
CP
spinal64
CP
!
2
mixing
time
CP
spinal64
dipolar CHHC experiment
(Lange et al., JACS 2002)
13C
CP
t1
CP
CP
t2
cross-polarization steps of short duration (100-200us)
1.
selectivity of the CP transfer -> unexpected cross-peaks
2.
3 fold rotation of CH3 prevent efficient transfer -> lack of methyl-methyl contacts
heteronuclear through-bond interaction
J-CHHC experiment
Carbon - carbons correlation which reflect proximities between their bonded H
Methyl Proton Contacts Obtained Using Heteronuclear Through-Bond Transfers in Solid-State NMR Spectroscopy
Loquet, Laage, Gardiennet, Elena, Emsley, Böckmann, Lesage
J. Am. Chem. Soc. 2008 130(32) 10625-10632
200
180
160
140
120
100
80
60
40
20
0
Carbon 13 chemical shift / ppm
Carbon 13 chemical shift / ppm
47Hδ-23Hγ
15
20
47Hδ-20Hβ
1
J-CHHC
CHHC
64Hδ-62Hγ
51Hε-19Hβ
78Hβ-6Hγ 54Hβ-55Hγ
44Hβ-77Hδ
19Hβ-14Hδ
26Hβ-33Hγ
44Hβ-63Hδ
78Hβ-74Hγ
44Hβ-35Hδ
61Hγ-81Hγ
61Hγ-35Hδ
2Hγ-74Hγ
42Hγ-35Hδ
19Hβ-47Hδ
3
20Hβ-16Hβ
44Hβ-26Hβ
73Hβ-33Hγ
21Hδ-20Hβ
33Hγ-44Hβ
12Hγ-55Hγ
77Hδ-26Hβ
33Hγ-63Hδ
55Hγ-14Hδ
81Hγ-35Hδ
23Hγ-47Hγ
2
16Hβ-51Hε
NHHC + CHHC
precision: 1.5 Å
accuracy: 1.8 Å
57Hγ-12Hγ
63Hδ-77Hδ
74Hδ-2Hγ
14Hδ-12Hγ
14Hδ-81Hγ
25
35Hδ-6Hγ
15
25
20
Carbon 13 chemical shift / ppm
30
1
*
*
2
*
*
3
**
*
35 30 25 20 15 10 35 30 25 20 15 10 35 30 25 20 15 10
13C chemical shift / ppm 13C chemical shift / ppm 13C chemical shift / ppm
NHHC + CHHC + J-CHHC
precision: 1.3 Å
accuracy: 1.1 Å
1H
CP
TPPM
C.W.
TPPM
highly reduce dipolar truncation
13C
CP
t1
C.W.
t2
PAR
Proton Assisted Recoupling and Protein Structure Determination
De Paëpe, Lewandowski, Loquet, Böckmann, Griffin.
JCP, accepted
promote long distance contacts
DARR 200ms
PAR 15ms
20
Carbon 13 chemical shift / ppm
Carbon 13 chemical shift / ppm
20
CHHC 200us
30
40
50
30
40
50
60
60
70
70
60
40
20
Carbon 13 chemical shift / ppm
60
40
20
Carbon 13 chemical shift / ppm
PAHR 15ms
PAHR 2ms
Carbon 13 chemical shift / ppm
20
20kHz
@ 900 MHz
Crh protein
30
40
50
60
70
60
40
Carbon 13 chemical shift / ppm
20
De Paëpe, Lewandowski, Loquet, Böckmann, Griffin,
JCP, accepted
. High-resolution structure from solid-state NMR data
deal with high-level of ambiguity
CHHC / NHHC experiments for distance restraints
. Additional restraints
J-CHHC
. 13C-13C correlation with efficient recoupling
PAR
Thanks
IBCP (CNRS UMR 5086)
Max-Planck-Institute
for Biophysical Chemistry
Massachussets Institute
of Technology
Anja Böckmann
Marc Baldus
Manuel Etzkorn
Henrike Heise
Adam Lange
Bob Griffin
Gael De Paëpe
Josef Lewandowski
Institut Pasteur
ENS Lyon
Michael Nilges
Thérèse Malliavin
Benjamin Bardiaux
Anne Lesage
Lyndon Emsley
Ségolène Laage
Carole Gardiennet
LBRS